CONCLUSION

From the experiment that we have done, finally, we can conclude that Gram staining

is the method of distinguishing between Gram-positive and Gram-negative bacteria. Gram-

positive bacteria have a thicker peptidoglycan which makes up their cell walls but have

greater lipid content since these bacteria have an outer membrane. The differences in their

cell wall architecture cause them to look different from one another by performing Gram

staining procedure.

Escherichia coli appeared reddish pink which indicate that these bacteria do not bind

the crystal violet stain. Therefore, it is determined to be a Gram-negative bacteria. The

decolourizer extracted the lipid from outer membrane of Gram-negative bacteria washed off

the crystal violet-iodine complex. Safranin then was used to counterstain Escherichia coli

leaving the colour pink.

Staphylococcus aureus absorbed the purple colour from crystal violet. This indicates

Staphylococcus aureus is Gram-positive bacteria which have thicker peptidoglycan in their

cell wall that trap the crystal violet-iodine complex more effectively, making them resistant to

the decolourizer.

In this experiment, we also have provided some material to help us for reaching the

aim of this experiment such as Hucker’s crystal violet, Gram iodine, 95 % ethyl alcohol,

safranin, and microscopic slide, staining rack and bibulous paper.

However, before doing the experiment, we absolutely need to pay attention on the

precautions. There are several procedures that we have to do in order to avoid the error in this

experiment, such as prepare smear from cultures of microorganisms, heat fix smears, place

slides on a staining rack and so on.

REFERENCES

1. Leboffe M.Pierce BE. 2010 .Gram Stain, P. 105-109. In Microbiology Lab Theory

and Application, 3rd ed., Morton Publishing CO., Englewood, Co.

2. Cappucino G.James, Sherman Nataline, 7th edition, Pearson Education.

3.
QUESTIONS

1. If the iodine step were omitted what colour would expect a Gram-negative

microorganisms to be? A Gram-positive?

♪ If the iodine in the Gram’s stain was omitted, the crystal violet enters the cell will

not able to form the crystal violet iodine complex. The crystal violet iodine

complex molecule has a larger size than that of crystal violet molecule and hence

Gram-positive bacteria are able to retain stain. If the iodine were omitted even

Gram-negative cells would get decolourized by alcohol treatment and would be

seen as a Gram-negative cells. Other than that, they would all be the colour of

your counterstain (usually safranin) because without the aid of Gram’s iodine,

which acts as a mordant (helps to set the dye into the organisms). Crystal violet

would not effectively set into the organisms, and when the decolourizer is added,

the crystal violet will be washed away and the counterstain will be the only stain

effecting the organisms.

2. Could other dyes be substituted for crystal violet Gram stain procedure? For safranin?

Explain your answer.

♪ Crystal violet dye can be replaced with methylene blue. This is because both are

basic dye that have a positive charge that able to bind to negatively charge

particle, such as the teichoic acid at the bacterial cell wall. Therefore, methylene
 blue can be used to replace crystal violet dye. Meanwhile, for safranin, it can be

replaced with basic fuchsin. Both dyes are able to stain Gram-negative bacteria

which is a pink colour but based on the information that we have searched, basic

fuchsin give much brighter colour in order for the bacteria to be seen clearly.

3. What role does the mordant play in the Gram stain procedure?

♪ The mordant is the substance that set dyes the iodine in Gram stain fixed the

crystal violet to bacterial cell wall of the Gram-positive bacteria. When Gram-

positive bacteria are treated with ethanol, the alcohol is thought to shrink the pores

of thick peptidoglycan. Thus, the dye-iodine complex is retained during this short

decolourization step and the bacteria remains purple.

4. What correlation is there between cell wall composition and Gram reaction? Is Gram

reaction correlated with sensitivity too various antibacterial agents?

♪ Crystal violet dissociate into Cl- ions and CV+ which interact with negatively

charged components of the stain in purple. Iodide ion (I-) is than react with CV+,

forming CV-I complex. Alcohol interacts with the lipid of cell membrane, causing

Gram-negative cell lose its outer lipopolysaccharides membrane, exposing the

peptidoglycan layer. Therefore, Cl-I and the outer membrane is washed away.

However, in the Gram-positive cell, the alcohol dehydrated the cell, trapping the

Cl-I because of the thick peptidoglycan. The Gram reaction is somehow correlated

with sensitivity to various antibacterial agents. Gram-positive cells are more

susceptible to mechanical breakage as the amount of peptidoglycan is small.

However, the reaction does not ensure that the same antibacterial agent is able to

destroy the two (2) different species or strain of bacteria even though they showed
the same result in the Gram reaction. This is because types of action such as injury

to the plasma membrane, inhibition of cell wall synthesis and prootein synthesis,

does not only depend on the Gram reaction.